This project seeks to understand how microbial antigens that are selectively expressed in vivo, and in specific tissues, contribute to the genesis of protective host immune responses and can serve as rational targets for vaccines. Borrelia burgdorferi has been shown to preferentially express different genes during infection of the mammalian host and within distinct host tissues. Moreover, B. burgdorferi has a tropism for the skin, joints, heart and nervous system, and these organs/tissues are associated with particular clinical manifestations of Lyme disease, such as erythema migrans or Lyme arthritis. B. burgdorferi genes that are selectively expressed in vivo and in precise host tissues will be identified using 2 strategies. First, differential immunoscreening, a technique developed in our laboratory, will be used. In this approach, a B. burgdorferi expression library is probed with 2 sets of sera (for example, [a] sera from an infected host, and [b] sera from a host hyperimmunized with killed B. burgdorferi) to identify antigens that only react with [a], and may therefore be selectively expressed in vivo. Secondly, DECAL (Differential Expression analysis using a Custom Amplified Library), will be adapted to identify B. burgdorferi genes expressed in the host. In this strategy, prokaryotic ribosomal RNA is removed for a bacterial expression library, which can then be used for subtractive analysis of gene expression in host tissues. Then the immune responses to the host-specific B. burgdorferi antigens will be characterized. We will directly assess tissue-specific gene expression, by RT-PCR using patient specimens and also in an experimental murine model of Lyme borreliosis. Then we will correlate antigen-specific immune responses with the course of Lyme disease in patients and determine whether immune responses to tissue-specific antigens influence the course of infection and/or prevent specific clinical manifestations of disease.